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J. Biol. Chem., Vol. 275, Issue 37, 28866-28872, September 15, 2000

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The Major Chicken Egg Envelope Protein ZP1 Is Different from ZPB and Is Synthesized in the Liver^*

Nina Bausek, Marianne Waclawek, Wolfgang J. Schneider, and Franz Wohlrab^§

From the Institute of Medical Biochemistry, Department of Molecular Genetics, University and Biocenter Vienna, Dr. Bohr-Gasse 9/2, A-1030 Wien, Austria

Received for publication, June 7, 2000, and in revised form, June 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The extracellular matrix surrounding vertebrate oocytes is called the zona pellucida in mammals and perivitelline membrane (pvm) in birds. We have analyzed this structure in chicken follicles and laid eggs and have identified a 95-kDa component of the pvm, which, by protein sequencing, shows homology to mammalian zona pellucida proteins. Surprisingly, we could not detect this protein in ovarian granulosa cells or oocytes but instead found high levels in the liver of the laying hen. In contrast, it is absent in rooster liver but can be efficiently induced by estrogen treatment of the animal. An immunoscreen of a liver -ZAP library yielded a cDNA coding for a protein of 934 amino acids. It displayed significant homology to members of the ZP1/ZPB family from other species, notably to mouse and rat ZP1, and was therefore designated chkZP1. It is clearly different from a protein designated chkZPB that had been deposited in the data base previously. Alignment of the known members of the ZP1/ZPB family demonstrated the existence of at least three subgroups, with representatives of both the ZP1 and the ZPB sequence homology group occurring in vertebrates. Northern blot analysis of liver extracts revealed the presence of a single 3.2-kilobase mRNA coding for chkZP1, distinct from the chkZPB transcript detectable in follicles. Immunohistochemical analysis of follicle sections demonstrates that chkZP1 can be found in the blood vessels of the theca cell layer as well as in the pvm surrounding the oocyte. Thus, in the chicken, at least one of the major pvm components is synthesized in the liver and is transported via the bloodstream to the follicle.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vertebrate eggs are surrounded by an insoluble extracellular matrix, which is called zona pellucida in mammals, chorion in fish, and perivitelline membrane or vitelline envelope in amphibians and birds (1-4). This structure represents the initial sperm-binding site, participates in the induction of the acrosome reaction, and mediates the prevention of polyspermy. In mammals, the zona pellucida is composed of three component glycoproteins, called ZP1, ZP2, and ZP3, (also known as ZPB, ZPA, and ZPC, respectively) (5), that show significant conservation across all species studied. The classification of these proteins often has been difficult because of their extensive heterogeneity, which is due to multiple posttranslational modifications. In many cases it has been possible to make unambiguous assignments to particular gene families only after isolation of the cDNAs.

Although the different polypeptides are highly conserved, they show large differences in their functional properties (6). Thus, the primary sperm receptor in mouse appears to be ZP3 through its O-linked oligosaccharides (7), whereas in the rabbit the ZP1 homologue (8), and in the pig, a heterodimer between ZPB (ZP1) and ZPC (ZP3), is thought to possess sperm binding activity (9). Furthermore, sperm binding in the pig appears to be mediated not by O-linked but rather by N-linked carbohydrates (10, 11).

Oocytes of oviparous species are large when compared with those of mammals. In the chicken, the pvm¹ is composed of two layers, an inner layer, deposited in the preovulatory phase, and an outer layer, added during passage through the oviduct; these layers are separated by a thin membrane (2). This membrane and the outer layer are added to the inner membrane only after ovulation, i.e. during migration of the oocyte through the oviduct (12, 13). For successful fertilization to occur, sperm first has to bind to the pvm, a process that is species-specific (14), and then penetrate it (15). The outer layer appears to be involved in a block to polyspermy via the acrosome reaction (16).

Despite the long history of ultrastructural studies on the chicken follicle (2), little is known about the molecular details of the composition of the pvm. Of the major bands obtained on SDS-polyacrylamide gels after electrophoresis of laid egg pvm under reducing conditions, all but two are outer layer components. Of these two, one protein of 34 kDa has been characterized (17, 18). This protein is a homologue of the mammalian ZP3/ZPC; it has been demonstrated to be synthesized exclusively by the granulosa cells surrounding the oocyte and is secreted in a polarized fashion (17). The nature of the other band had been unclear. In this paper, we report the characterization, molecular cloning, site of expression, and localization of this protein and its identification as an avian ZP1 homologue. We also show that it is clearly different from a related protein designated chkZPB.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals-- 30-40-week-old Derco-brown laying hens and roosters (Heindl Co., Vienna, Austria) were used as a source for eggs, follicles, and tissues. Antibodies were raised in adult female New Zealand White rabbits (see below).

Preparation and Solubilization of Chicken pvm-- Perivitelline membranes from chicken eggs or ovarian follicles were obtained as described (17). Briefly, for isolation of pvm from freshly laid eggs, yolk was drained from oocytes by puncturing the membranes after removal of egg white. The membranes were washed in Tris-buffered saline (137 mM NaCl, 2.5 mM KCl, 2.5 mM Tris·HCl, pH 7.6) and incubated for 1 h at 4 °C in 200 mM Tris·maleate (pH 6.5), 2 mM CaCl₂, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 mM leupeptin, and 1.4% Triton X-100. After centrifugation for 40 min at 4 °C at 300,000 × g, the detergent-pretreated membranes were solubilized in the equivalent of 0.2 ml/egg of Tris-buffered saline containing 2% SDS and 50 mM dithiothreitol. Insoluble constituents were removed by centrifugation at 12,000 rpm for 20 min in an Eppendorf centrifuge.

For isolation of pvm from follicles, ovaries were dissected from mature laying hens immediately after decapitation. Granulosa cell sheets containing the inner pvm as well as granulosa cells and basement membranes were prepared from follicles larger than 2 cm in diameter as described (19). The protocol for subsequent solubilization was identical to the one for pvm from laid eggs.

Protein Sequencing and Immunological Procedures-- Chicken pvm proteins were separated by preparative SDS-PAGE (4.5-18%) under reducing conditions. The 95-kDa band was cut out and eluted electrophoretically at 180 V for 8 h in 25 mM Tris, 250 mM glycine, pH 8.3, containing 0.1% SDS. Microsequencing of tryptic digests was performed essentially as described (20). Three sequence fragments were characterized, designated 95.1 (the N-terminal peptide, LLQYHYDCRDFGMQLLAYP), t2 (TQLVPVGPATLQLPF), and t3.1 (PGLXXPGLPSXPGLVS), respectively. A 190-kDa band present under nonreducing conditions (see Fig. 2) had the same N-terminal sequence as the 95-kDa species and is presumably a dimeric form of this protein.

Subsequently, synthetic peptides were obtained corresponding to fragments 95.1 and t2, coupled to maleimide-activated keyhole limpet hemocyanin (Pierce), and used to raise antisera in rabbits. Three series of intradermal injections of 500 µg of each of the antigens in a total volume of 400 µl, mixed with an equal volume of Freund's complete (day 0) or incomplete (days 14 and 35) adjuvant were administered to the animals. Additionally, an antiserum directed against the entire gel-purified 95-kDa protein (anti-p95 antiserum) was raised according to the same protocol. Preimmune and immune sera were stored at 80 °C.

Cloning and Sequencing of Chicken ZP1-- A laying hen liver  ZAPII cDNA library (Takara) was grown and induced with isopropyl-1-thio--D-galactopyranoside-soaked filters. 1 × 10⁶ plaques were screened with polyclonal anti-p95 antiserum (see above) according to standard methods (21) as modified by the picoBlue^TM immunoscreening kit (Stratagene). Four positive clones were picked, and the released phage particles were grown in XL1-Blue MRF' cells that were coinfected with ExAssist^TM helper phage for in vivo excision using the ExAssist/SOLR system (Stratagene).

All clones obtained were sequenced on both strands. The phage were digested with several restriction enzymes and recloned into BlueScript vectors, and the contig sequences were determined on an ABI sequencer. Contigs were assembled with the help of Assembly Line (Oxford Molecular) and alignments were determined by ClustalW (MacVector 6.5.3).

Northern Blotting-- Chickens were sacrificed by decapitation, and tissues were frozen immediately in liquid nitrogen. For estrogen treatment, roosters were injected intramuscularly with 10 mg/kg 17--estradiol (dissolved in 1,2-propanediol at 20 mg/ml) 48 h prior to removal of tissues; control roosters received vehicle only. Total RNA was extracted using TriReagent (Molecular Research Center, Inc.) and was subjected to electrophoresis on a 1.2% agarose gel in the presence of glyoxal (22), followed by blotting onto positively charged nylon membranes (Roche Molecular Biochemical). RNA was covalently bound to the dried membrane by UV cross-linking. A 2921-base pair fragment of chkZP1 was used as hybridization probe and labeled with [-³²P]dCTP by random priming. Hybridization was performed at a probe concentration of 2 × 10⁶ cpm/ml in a buffer containing 1% bovine serum albumin, 7% SDS, 0.5 M sodium phosphate (pH 6.8), and 1 mM EDTA at 65 °C for 16 h. Washes were performed at 65 °C, first in 40 mM sodium phosphate buffer (pH 6.8), 0.5% bovine serum albumin, 5% SDS, and 1 mM EDTA and then in 1% SDS, 40 mM sodium phosphate (pH 6.8), and 1 mM EDTA. The blot was subsequently exposed to X-Omat Blue XB-1 (Kodak) film with intensifying screen at 80 °C.

PCR Amplification of a Chicken ZPB-specific Probe-- To obtain a chicken ZPB (accession number AB025428)-specific probe, total RNA was isolated from small follicles using TriReagent (Molecular Research Center, Inc.), according to the manufacturer's instructions. 5 µg of this RNA was then reverse transcribed using the Superscript^TM preamplification system (Life Technologies, Inc.), and an aliquot of the obtained cDNA was used as a template for PCR. Primers were chosen to amplify a 510-base pair region of the chicken ZPB cDNA (forward primer, 5'-TTGGAGCTGTGTTCTTCTTGG-3'; reverse primer, 5'-GGTTGTAACAACAGCCTCGC-3'). PCR was performed for 30 cycles of denaturation for 1 min at 95 °C, annealing was performed for 1 min at 58 °C, and primer extension was performed for 1.5 min at 72 °C. The PCR product was subcloned into pCR2.1 (Invitrogen), and its identity was verified by sequencing. It was then labeled with [-³²P]dCTP by random priming and used as a hybridization probe for Northern blotting as described above.

Western Blotting-- Freshly obtained chicken tissues were homogenized in 5 ml/g of buffer A (25 mM Tris·HCl, pH 8.0, 1 mM CaCl₂, 1 mM phenylmethylsulfonyl fluoride, and 1 µM leupeptin) using an Ultra Turrax T25 and then centrifuged at 1,500 × g for 10 min. The supernatant was passed through cheesecloth and spun at 100,000 × g for 1 h. The resulting pellet was resuspended in buffer A and aspirated through 18- and 22-gauge needles. After recentrifugation, cells were resuspended in extraction buffer (125 mM Tris·maleate, pH 6.0, 1 mM CaCl₂, 0.5 mM phenylmethylsulfonyl fluoride, 1 µM leupetin, 160 mM NaCl, and 1% Triton X-100).

Serum was delipidated prior to gel electrophoresis by treatment with a 20-fold excess of precooled chloroform/methanol (2:1). After 30 min at 4 °C, the mixture was spun for 15 min at 4000 rpm in a tabletop centrifuge, followed by dissolution of the pellet in the original volume of sample buffer.

Protein extracts and sera were separated by one-dimensional SDS-PAGE (23). Proteins were transferred to nitrocellulose (Hybond-ECL, Amersham Pharmacia Biotech) for immunoblotting. Transfers were performed in 25 mM Tris, 192 mM glycine, and 20% methanol for 1 h at 17 V at room temperature or overnight at 6 V and 4 °C. The membranes were blocked in 80 mM Na₂HPO₄, 20 mM NaH₂PO₄, 100 mM NaCl (PBS), 0.1% Tween, 5% nonfat dry milk for 1 h, followed by incubation with antiserum in PBS-t (PBS with 0.1% Tween). After three washes in PBS-t, the membrane was incubated with protein A-horseradish peroxidase (1:5000) for 1 h. Bands were visualized by the enhanced chemoluminescence procedure as suggested by the manufacturer (Amersham Pharmacia Biotech). The positions of migration of molecular weight standards (Bio-Rad) were determined by staining with Ponceau S (0.5% in 1% acetic acid).

Glycosidase Digestions of pvm-- Perivitelline membranes from follicles were obtained as described above, washed in Tris-buffered saline, and taken up in 20 mM sodium phosphate (pH 6.8), 10 mM EDTA, 0.2% SDS. After addition of 1 milliunit of N-glycosidase F (Roche Molecular Biochemicals; from Flavobacterium meningosepticum)/100 µl of suspension and subsequent incubation at 37 °C for 16 h, the samples were centrifuged at 15,000 × g for 2 min, and the supernatant was analyzed by SDS-PAGE as described.

Immunohistochemistry-- For differential interference contrast microscopical analysis of tissue sections after immunohistochemistry, procedures were as described (24). Briefly, chickens were anesthetized with 2 ml of nembutal and perfused via the left ventricle with 300 ml of PBS, followed by 300 ml of a solution containing 75 mM L-lysine, 75 mM sodium phosphate (pH 7.3), 2% paraformaldehyde, and 2.4 mg/ml sodium meta-periodate. Specimens were embedded in paraffine using a Tissue-Tek VIP (Miles Scientific) embedding machine, and 3-µm slices were cut on a Microm HM335E microtome. Slices were deparaffinized in xylol exchange medium XEM-200 (Vogel) and rehydrated by consecutive washes in 96, 70, and 50% ethanol and pure water. Endogenous peroxidase was blocked by incubating the slices in 3% H₂O₂ for 5 min. Unspecific binding of antibodies was inhibited by blocking with a solution of 1% milk powder and 3% total goat serum in PBS for 1 h at room temperature. Polyclonal anti-p95 antiserum was applied at a dilution of 1: 800 in PBS for 1 h. After three washes in PBS, the following incubations were performed at room temperature: goat anti-rabbit biotinylated IgG (Sigma) diluted 1:500 in blocking solution for 1 h, five washes with PBS, peroxidase-labeled avidin (Sigma; 1:200 in 1% milk in PBS) for 1 h, and three final washes with PBS. For the color reaction, slices were incubated in 0.1 M sodium acetate (pH 5.1) containing 150 µl of 30% H₂O₂ and 20 mg of 3-amino 9-ethylcarbazole/100 ml of buffer. The staining process was followed under the microscope and stopped by immersing the slides in water. Results were observed on a Zeiss Axiovert 135 microscope.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Biochemical Characterization of a 95-kDa Chicken Egg Envelope Component-- In a previous communication, we had demonstrated that SDS-dissolved chicken perivitelline membranes from laid eggs, when subjected to SDS-PAGE under reducing conditions, show four major protein bands with apparent molecular weights of 5,000, 13,000, 34,000, and 95,000, respectively (17). Sequencing of the N termini and of tryptic fragments of the proteins indicated that the 5- and 13-kDa species represent chicken vitelline membrane outer protein I and lysozyme, respectively, whereas the 34-kDa protein is an avian homologue of the mammalian zona pellucida component known as ZP3 or ZPC (17). The nature of the 95-kDa protein (p95) was not immediately clear. Although BLAST searches of public data bases revealed that the N-terminal sequence had homology to proteins of the ZP1/ZPB family, an internal tryptic fragment (t3.1) showed no similarity to known zona pellucida components of other species. Rather, it resembles glutamine-, proline-, and glycine-rich proteins such as fibroin or glutenin, so that an unambiguous assignment to the zona pellucida protein family was not directly possible. Furthermore, during the course of this work, a chicken ZPB sequence had been deposited in the public data bases (accession number AB025428); however, it did not contain any of the fragments we had sequenced.

To characterize the 95-kDa band, we first raised polyclonal antisera against the entire protein as well as against synthetic oligopeptides corresponding to the sequences we had obtained (see "Materials and Methods"). Fig. 1 shows identical samples run on the same gel probed with three different antibodies. As is evident in panel A, the antiserum against the entire protein (lane 1) as well as the one against the N-terminal peptide 95.1 (lane 2) recognize only a single band of the expected size on immunoblots of chicken pvm. Although the same polypeptide is recognized by the antiserum against the internal peptide t2, we consistently also observed reactivity toward two polypeptides of 43 and 48 kDa, respectively (lane 3). The nature of these bands is unclear; however, Coomassie Blue-stained SDS-polyacrylamide gels of pvm extracts show the 95-kDa protein as well as the 34-kDa ZP3 but do not exhibit major proteins migrating at these positions (17).

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Immunoblotting of p95. A, extracts of chicken pvm (10 µg of protein/lane) prepared from the second largest follicle (diameter, ~2.5 cm) were subjected to SDS-PAGE (12%) under reducing conditions. The proteins were transferred to nitrocellulose and immunodetected with antibodies against the entire p95 protein (lane 1), against a peptide (95.1) representing the N terminus of the mature protein (lane 2) and against an internal peptide (t2; lane 3) as described under "Materials and Methods." The positions of migration of molecular mass standards (Life Technologies, Inc.) are indicated. B, laying hen pvm extracts (10 µg/lane) were subjected to SDS-PAGE (10%) under reducing conditions before (lane 1) or after (lane 2) treatment with N-glycosidase F as described under "Materials and Methods." Immunodetection was performed with the antibody directed against the entire p95 protein.

To test for the possibility that p95 is a glycoprotein, the pvm was treated with N-glycosidase F and subsequently subjected to SDS-PAGE. As can be seen in Fig. 1B, the band detected by the antibody directed against the entire protein was shifted to an apparent molecular mass of approximately 93 kDa (lane 2). This increase in mobility indicates the presence of N-linked sugars on p95.

Hepatic Synthesis of p95-- Surprisingly, the pvm is not the only tissue where p95 can be detected. As can be seen in Fig. 2, it is also present at high levels in the liver and serum of laying hens but is lacking from the liver and serum of roosters. Because for this immunoblot, SDS-PAGE had been performed under nonreducing conditions, an additional band migrating at approximately 190 kDa representing a dimeric form of p95 (17) was observed. In mammals and most other species studied to date, zona proteins are synthesized either in the oocyte itself or in the follicle cells surrounding it. In some species of fish, however, the liver has been reported to be the site of synthesis for components of the piscine equivalent of the zona pellucida. These proteins are then transported to the oocytes via the blood stream (25-29). The results of Fig. 2 suggest that a similar situation exists in the chicken, a finding that is unexpected in light of the site of synthesis of chicken ZP3, namely ovarian granulosa cells (17). Fig. 2 also demonstrates that p95 expression is sex-specific, because it is largely absent from rooster liver and rooster serum. These data strongly suggest that the liver is the site of synthesis for p95 and raise the possibility that the gene is under estrogen control. Indeed, estrogen treatment of roosters resulted in a dramatic induction of p95 synthesis in liver and serum to levels comparable with those in the laying hen (Fig. 2).

View larger version (29K): [in this window] [in a new window]   Fig. 2.   Estrogen dependence of p95 expression in liver and serum. Delipidated sera and Triton X-100 extracts from liver of laying hen (LH) and rooster (R) were prepared as described under "Materials and Methods." Estrogenized rooster (ER) tissues were obtained after treatment of male Derco Brown chickens with estradiol as outlined under "Materials and Methods." 4.5-18% SDS-PAGE of extracts (15 µg of protein/lane) was performed under nonreducing conditions. After transfer to nitrocellulose, bands were visualized with the anti-p95 antibody as described under "Materials and Methods."

Cloning of a Chicken ZP1 Homologue-- The notion that p95 might be synthesized by hepatic tissues prompted us to screen a -ZAP laying hen liver cDNA library with our antiserum raised against purified p95. We obtained several candidate clones, which were sequenced on both strands (see "Materials and Methods"). All clones were overlapping, apparently representing different parts of a single transcript of 2932 nucleotides in length. The sequence has been deposited in the public data bases under accession number AJ289697. It contains a long open reading frame coding for a polypeptide of 934 amino acids with a predicted molecular mass of 100 kDa (Fig. 3A). Comparison of the sequence of this protein with the sequence of the N-terminal peptide (95.1; see above) indicates that Leu-25 of the precursor protein represents the N terminus of the mature protein found in the laid egg. In fact, theoretical calculations using the algorithm of von Heijne and co-workers (30) predict a signal peptide with the most likely cleavage site at the sequence GLALL at exactly this position. For this reason and on the grounds of sequence alignments, we believe that the first ATG in the cDNA (position 9) indeed represents the initiator codon. In addition, we find a consensus furin cleavage site (RX(K/R)R) at position 900-903. The protein contains 21 cysteines, as well as three consensus sites for N-linked glycosylation (Asn in positions 65, 121, and 723). The putative mature protein lacking the signal sequence and the C terminus would have a molecular mass of 94 kDa, in excellent agreement with the results obtained by immunoblotting (Fig. 1).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Amino acid sequence of chicken ZP1. A, conceptual translation of the sequence of the cDNA obtained by immunoscreening a laying hen liver library. Peptides obtained by microsequencing are printed in bold italic letters, and an asterisk indicates the N terminus of the mature protein. The zona pellucida (ZP) domain (solid underlining) and the trefoil domain (dotted underlining) are marked. Putative N-glycosylation sites are highlighted in bold letters, and a furin consensus sequence is double underlined. B, sequence relationships of several members of the ZP1/ZPB family are shown by a dendrogram created by MacVector 6.5.3. Sources and accession numbers are as follows: medaka choriogenin H, D89609; winter flounder ZP protein, U03674; salmon eggshell protein clone ZP23, AJ000665; mouse ZP1, U20448; rat ZP1, AB000928; human ZP1, extracted from PAC clone AC004126 according to Ref. 49; chicken ZP1, AJ289697; Macaca radiata ZP-1, CAA71410; human ZPB, U05781; marmoset ZP1, Y10827; rabbit 55 K ZPB, Q00193; cat ZPB, S70400; chicken ZPB, AB025478; and Xenopus laevis ZPB, U49950.

Comparative analysis of the protein sequence with the data bases using the BLAST algorithm revealed high scoring matches with the ZP1/ZPB family of proteins. Alignment of the chicken sequence with ZP1/ZPB proteins from other organisms shows a highly conserved hydrophobic region, called the zona pellucida domain (31), in the C-terminal part of the molecule. Further similarities are the presence of a so-called trefoil domain (32) and the above-mentioned furin cleavage site. In addition, the extreme N terminus also has significant homology to some other ZP1/ZPB family members, notably to those found in mouse and rat. In contrast, the central part of the p95 sequence, which contains one of the tryptic fragments (t3.1) that we had sequenced initially, shows no apparent similarity to any of the mammalian zona pellucida components. Taken together, the alignment demonstrates that the chicken isolate is highly homologous to ZP1/ZPB proteins in both the N- and the C-terminal domains but contains an additional central region with some resemblance to several other extracellular proteins like fibroin or wheat glutenin. The similarity of p95 to other zona proteins and the fact that we had originally isolated it as one of two major components of the inner pvm support its assignment to the family of zona pellucida proteins.

ChkZP1 Is Different from chkZPB-- The sequence data also clearly distinguish this protein from a sequence deposited in the data base called chicken ZPB. In fact, the dendrogram in Fig. 3B shows that the sequence obtained here appears to be more closely related to the mouse, rat, and human ZP1 proteins than to the chicken or human ZPB polypeptides. This indicates that it represents a distinct avian gene related to the ZP1 family, and we therefore designate it chicken zona pellucida protein 1 or chkZP1. The dendrogram further suggests that, according to sequence homologies, we can distinguish between several distinct subgroups in the ZP1/ZPB gene family. One group comprises the mouse, rat, human, and chicken ZP1 proteins (ZP1 group), one contains the human, rabbit, chicken, cat, and Xenopus ZPB proteins, as well as the marmoset and macaque ZP1 orthologues (ZPB group), and a third group encompasses the fish homologues from winter flounder, medaka, and Atlantic salmon (Fig. 3B).

Expression of chkZP1-- Using a probe spanning the entire coding region of the cDNA, we then performed Northern blotting experiments on various chicken tissues (Fig. 4). It is obvious that the only major site of synthesis of chkZP1 mRNA is the liver. Some transcripts are also detectable in the adrenal glands, but at low levels when compared with hepatic tissues (Fig. 4A). The probe hybridizes to a single transcript of approximately 3.2 kilobases, suggesting that major splice variants are not expressed in the tissues studied. In accordance with the results obtained by immunoblotting (Fig. 2), the chkZP1 transcript is absent in rooster liver but can be induced by estrogen treatment of the animal to levels comparable with those detected in mature females (Fig. 4B). It is noteworthy that we could not detect significant expression in gonadal tissues, such as follicles (Fig. 4B) or rooster testes (Fig. 4A), even after prolonged exposure of the films to the blots (data not shown). This expression pattern resembles the one observed in teleost fish (25-29) but is in contrast to the one reported for other vertebrate ZP1/ZPB homologues (33-35).

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Expression pattern of chkZP1 mRNA. A, total RNA (10 µg) isolated from laying hen liver, kidney, adrenals, spleen, lung, heart, brain, muscle, and rooster (R) testis was denatured, separated by electrophoresis on a 1.2% agarose gel, and transferred to nylon membranes (Amersham Pharmacia Biotech). 32P-Labeled full-length chkZP1 cDNA was used as a probe (see "Materials and Methods"). The positions of the size markers (1-kilobase DNA ladder) are indicated on the left. B, total RNA from laying hen (LH) liver and follicle, as well as rooster liver harvested before (R liver) or after (ER liver) estrogen treatment, were prepared and analyzed as described above.

To investigate the relation between our isolate (accession number AJ289697), and the chicken ZPB sequence in the data base (accession number AB025478), we used a probe complementary to chkZPB (see "Materials and Methods") in Northern blots on extracts from chicken tissues. No hybridization to extracts from liver, blood, or follicles larger than 5 mm in diameter could be detected (Fig. 5), indicating different expression patterns for chkZP1 and chkZPB. The only signals were obtained from very small stroma-embedded follicles and from small white follicles (smaller than 1 mm in diameter); these RNAs migrated with an apparent size of approximately 2.5 kilobases. We also attempted reverse transcription-PCR from liver extracts using the chkZPB-specific primers described under "Materials and Methods" but failed to get any amplified product (data not shown). Taken together, the different transcript lengths (3.2 and 2.5 kilobases, respectively), the relatively weak sequence homology between them (23%), and the different sites of synthesis show that the two proteins are distinct gene products. The reasons for the existence of two ZP1/ZPB proteins, as well as their functions, are unclear.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Expression pattern of chkZPB mRNA. Total RNA (40 µg) was prepared from laying hen (LH) liver, the largest (F1) and fourth largest (F4) preovulatory follicles, small yellow (SY) follicles, large and small white (LW/SW) follicles, and very small stroma-embedded (Stroma) follicles, as well as from rooster (R) testis and was analyzed by Northern blotting as described under "Materials and Methods." A 32P-labeled fragment of chkZPB was used as a probe (see "Materials and Methods"). The positions of molecular size markers are indicated on the left.

Immunohistochemistry of chkZP1 in Follicles-- To ascertain the site of deposition of ZP1, we performed immunohistochemistry on sections from follicles harvested at the beginning of the rapid growth phase (diameter, ~4 mm) using the anti-ZP1 antiserum (Fig. 6). It can be clearly seen that chkZP1 is localized in the pvm and that it is absent in the neighboring granulosa cell sheet. The outer layers of the follicle cells, the theca externa, is heavily vascularized; here, we observe some positive staining, corroborating the finding that chkZP1 is present in high concentrations in the serum (Fig. 2). In the theca interna, where vascularization decreases, the amount of immunoreactive material is also reduced. ChkZP1 therefore appears to be synthesized in the liver and to be transported via the bloodstream to its target site, the pvm. Because it is blood-borne, chkZP1 can be detected in the many blood vessels within the stroma that embeds small developing oocytes (data not shown). A few 100-150-µm-diameter oocytes appear to be surrounded by a chkZP1-containing coat, whereas most others do not exhibit these structures. The fact that at this stage no chkZP3 is present around the developing oocyte (17) indicates that, in contrast to the mouse (36), the chicken zona pellucida proteins are expressed sequentially.

View larger version (123K): [in this window] [in a new window]   Fig. 6.   Immunohistochemical localization of chkZP1. Sections of a small follicle (diameter, ~4 mm) were prepared and processed for immunohistochemistry as described under "Materials and Methods." Treatment with anti-chkZP1 antibody clearly stains the perivitelline membrane (PVM) between the oocyte (OO) and the granulosa cell layer (GC). Some staining can be detected in the outer part of the theca layer (TC).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The inner layer of the pvm in hens is a large, three-dimensional extracellular network of fibers that is the functional homologue of the egg envelopes in other vertebrates, called vitelline envelope in amphibians and zona pellucida in mammals. In most animals, the egg envelope consists of three glycoproteins, called ZP1/ZPB, ZP2/ZPA, and ZP3/ZPC. In the pvm of the hen, only two components have been identified so far: a 34-kDa glycoprotein homologous to the mammalian ZP3/ZPC and a 190-kDa protein that, on SDS gels under reducing conditions, migrates as a 95-kDa species (17, 18). In this communication, we identify this protein as chkZP1, a sequence homologue of the vertebrate ZP1/ZPB family. Like the other members of the family, chkZP1 contains a zona pellucida domain, a motif of about 260 amino acids with eight conserved cysteines found not only in proteins of the egg envelope but also in other receptor-like glycoproteins (31). These include - and -tectorin, proteins of the inner ear (37-39), glycoprotein GP2, the major component of pancreatic secretory granule membranes (40), uromodulin (41, 42), and transforming growth factor- receptor type III (43). The signal sequence at the N-terminal end, a trefoil domain (32), and a furin-cleavage site (44) downstream from the zona pellucida domain (31) are also conserved. Whether furin cleavage actually occurs in vivo remains to be established.

Although the amino acid sequence similarity between chkZP1 and other ZP1 homologues is extensive, there are also some significant differences. The chicken protein contains a glutamine- and glycine-rich insert of approximately 230 amino acids not found in mammalian ZP1s in the N-terminal half of the molecule. This domain is organized into a number of partially overlapping and complex repeated sequences of variable length, including the sequence PGLQSQNQ. So far, such repeats in the N-terminal half of the protein have been reported to occur only in the ZP homologues of several fish species, such as winter flounder (27), medaka (29, 45), carp (46), gilthead sea bream (28), and zebrafish (47). It has been hypothesized that the function of these motifs is to assist in the hardening of the egg envelope after fertilization (47), but this theory remains speculative.

In all mammals studied so far, zona pellucida proteins are either synthesized by the oocyte itself or by the somatic tissues immediately surrounding it. ChkZP1 is made in the liver and possibly to a much lesser extent in adrenals and apparently is then transported through the blood stream to the follicles. Its expression is under estrogen control, and the fact that we can induce male chickens to produce ZP1 in the liver proves definitively that extragonadal tissues can be sites of synthesis of the protein. Similar expression patterns have only been observed in some species of teleosts. Thus, in rainbow trout (26), cod (25), winter flounder (27), sea bream (28), and medaka (29) egg envelope proteins derive from hepatic tissues and are expressed under the influence of estrogen. However, in these animals, the liver expresses all of the egg envelope proteins; in the chicken, the granulosa cell layer surrounding the oocyte produces ZP3 (17), whereas ZP1 is expressed in the liver. It remains to be seen whether the presence of the Pro-Glu-rich repeat domain in certain ZPs surrounding large oocytes is related to their extraovarian synthesis and/or to the mechanism of their transport to the follicle.

For the murine zona pellucida, a model has been proposed in which filaments of ZP2/ZP3 oligomers are cross-linked by dimers of ZP1, which is present at much lower levels than the other two components (48). Analysis of the inner layer of chicken pvm reveals the presence of only two major bands, corresponding to ZP1 and ZP3. There is no obvious homologue of the ZP2 family. It is unlikely that a putative ZP2 is masked by the other components of the pvm on SDS gels, because both the electrophoretically isolated ZP1 and ZP3 peptides were shown to be pure proteins by microsequencing. It is, however, possible that a putative ZP2 is much less abundant than ZP1 and ZP3 or that it is lost during pvm isolation and solubilization. In this context, it is interesting that the antiserum directed against peptide t2 shows additional bands upon Western blotting of pvm (Fig. 1). This peptide derives from within the zona pellucida domain of chkZP1 and could conceivably recognize additional zona proteins present at low concentrations. Studies to investigate this possibility are in progress.

Despite the apparent presence of only two major proteins in the inner pvm, the protein we describe here is the third zona pellucida protein homologue cloned from chicken tissues. In addition to the chkZP3 (17) and the chkZP1 described in this paper, the sequence of a cDNA specifying a protein called chicken ZPB has been deposited in the data base. The translation product of this cDNA has a theoretical molecular mass of 56 kDa, but it is presumably glycosylated and shows high homology to mammalian ZPB proteins. We have not found transcripts of the chicken ZPB gene in either hepatic tissues or large follicles but have been able to detect them by Northern blot analysis in small follicles (Fig. 5). Because, at present, antibodies directed against ZPB are not available, questions about the biosynthesis and function of this polypeptide cannot be answered yet.

Interestingly, a novel human zona pellucida gene (hszp1) has recently been identified on a cosmid (49). Its putative gene product is 67% identical to murine ZP1, whereas the identity between the previously described human ZPB and murine ZP1 proteins is only 33%. On the grounds of these and other data, Hughes and Barratt (49) have suggested that the true sperm receptor in vertebrates is a heterodimer between ZP3 and ZPB and not ZP3 alone. That ZP1 may have an entirely different function (49) is supported by the existence of separate ZPB and ZP1 genes in organisms as different as chicken and man. The results of a sequence comparison between a number of ZP1/ZPB proteins show the existence of three subgroups (Fig. 3). The chkZP1 clone is more closely related to ZP1 isolates from mouse, rat, and man, whereas the chkZPB protein, in contrast, resembles the ZPB isolates from marmoset, macaque, cat, rabbit, frog, and man. Whether distinct genes for ZP1 and ZPB homologues, respectively, exist in other species besides humans and chickens remains to be established.

	ACKNOWLEDGEMENTS

We thank Dr. T. Matsuda (Nagoya University) for discussions and information about the chicken ZPB sequence and Andreas Pecka for technical assistance.

	FOOTNOTES

^* This work was supported by Grant 309 from the Anton Dreher-Memorial Fund (to F. W.) and Grant F-608 from the Austrian Science Foundation (to W. J. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of a Studentship of the Vienna Biocenter International Ph.D. Program (Grant WK-001 of the Austrian Science Foundation).

^§ To whom correspondence should be addressed. Tel.: 43-1-4277-618-13; Fax: 43-1-4277-9618. E-mail: wohlrab@mol.univie.ac.at.

Published, JBC Papers in Press, July 3, 2000, DOI 10.1074/jbc.M004944200

	ABBREVIATIONS

The abbreviations used are: pvm, perivitelline membrane; PAGE, polyacrylamide gel electrophoresis; contig, group of overlapping clones; PCR, polymerase chain reaction; PBS, phosphate-buffered saline.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

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